Fig. 5. D-serine controls Arabidopsis
pollen tube growth in planta. (A) Effect of 100 mM D-Ser on Arabidopsis
wild-type pollen tube apical Ca2+
influx. Note the increase in oscillation amplitude. (B) b-glucuronidase
activity in mature pistils from transgenic plant lines transformed with
the serine-racemase promoter (1800
base pairs) cloned upstream of b-
glucuronidase. (C and D) D-Ser immunolocalization in wild-type and
sr1-1 pistils (D-Ser, blue; red is autofluorescence, merged for structural
correlation). (E) Insertion line with a mutation for serine-racemase (sr1-1). No serine-racemase transcript was detected
in insertion line. (Inset) RT-PCR on cDNA from flower. Images show callose staining on sr1-1 pistils pollinated
with wild-type pollen. Note balloon-like tip and branched pollen tubes, which are not observed in wild type.
grown in planta (Figs. 3C and 5D and fig. S8B).
These results suggest that D-Ser formed in the
pistil may have a subsidiary role in the navigation
of pollen tubes by modulation of GLRs.
Discussion. Our results show that D-Ser ac-
tivates GLRs in the apical region of pollen tubes,
allowing Ca2+ permeation into the cytoplasm,
thereby shaping the Ca2+ signature by modula-
tion of both Ca2+ influx intensity and oscillation
amplitudes. D-Ser concentration was measured in
the mM range in plant extracts (31, 32), and immu-
nolocalization results show strong concentration
differences in plant tissues, making it plausible
that it may reach concentrations within the range
we used in our in vitro experiments. However, we
cannot overrule other effects of serine racemase,
the latter having 20 times as much dehydratase
activity as racemase activity (29). Animal GLRs
play important roles in fast excitatory neurotrans-
mission in the central nervous system. They are
involved in neuron development as well as in neu-
ron plasticity and participate in integrated cognitive
processes such as memory and learning (33). The
data we now report reveal conservation of an amino
acid–based signal transduction involving oscilla-
tions, where similar channels perform their role by
affecting specific kinetic properties of Ca2+-induced
neurotransmitter release. The previous demonstra-
tion of another amino acid (g-aminobutyric acid)–
ionotropic receptor (34) pair involved in pollen-pistil
interaction makes an interesting parallel to the
data we now present, suggestive of a much wider
role of these kinds of mechanisms in cell-cell com-
munication of plant tissue and organs.
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Acknowledgments: E.M. acknowledges Fundação para a
Ciência e a Tecnologia (SFRH/BPD 21056/2004),
European Molecular Biology Organization (ASTF 193-2007),
and Agropolis fellowships. We thank M. Watahiki
(University of Hokkaido) for the YC3.1 construct and
M. Iwano (Nara Institute of Science and Technology,
Japan) for the YC3.6C Arabidopsis line. glr1.2-1
and glr3.7-1 insertion lines were originally characterized
by P. Walch-Liu, X. Q. Qu, and Y. Y. Gong in L.-H.L.’s
laboratory, in collaboration with M. Tester (University of
Adelaide) and B. Forde (University of Lancaster). We thank
N. Moreno and P. Almada for imaging support, A. Sommer
for protoplast preparation, C. E. Tadokoro for immunofluorescence advice, J. Becker for helpful discussions on the
genetic design of the experiments, P. Dias for help in
oscillation analysis, and J. B. Thibaud (Institut National de la
Recherche Agronomique-Supagro, Montpellier) for other
institutional support to E.M. J.A.F.’s laboratory is supported by
the Centro de Biologia do Desenvolvimento (FCT U664)
and FCT grants BIA-BCM/108044/2008 and QUI/64339/2006.
G.O.’s laboratory is supported by Austrian Science Fund
grant P21298. L.-H.L. was supported by China National
Science Foundation grant 30771288.
Supporting Online Material
Materials and Methods
Figs. S1 to S9
1 December 2010; accepted 4 March 2011
Published online 17 March 2011;